Synthesis, Biological Evaluation and Docking Analysis of Some Novel Quinazolin Derivatives as Antitumor Agents

Different acid chlorides (2a-d) reacted with anthranilic acid to produce 2-substituted-3, 1-benzoxazin-4-one (3a-d) which was used as starting material to synthesize some condensed and non-condensed heterocyclic compounds by reaction with nitrogen nucleophiles e.g., hydrazine hydrate, and formamide. Some of the newly synthesized analogues were chosen to evaluate their cytotoxic activity against human carcinoma cell lines (HePG2– MCF7– A549). The docking and the cytotoxic activity results revealed that nearly all of the compounds containing N-phenyl aniline showed signiﬁcant inhibition for the three cell lines.

Benzoxazine heterocyclic compounds are potent non-steroidal progesterone receptor agonists (33) having many other activities such as anticancer, antiangiogenic (34), antidiabetic and hypolipidemic (35), antidepressant (36) and antiplatelet aggregation activities (37). Epidermal growth factor receptor (EGFR), which is cellular trans-membrane tyrosine kinase, is over-expressed in a significant number of human tumors (e.g., breast, ovarian, colon and prostate). An EGFR expression level often correlates with vascularity and is associated with poor prognosis in patients. Inhibitors of the EGFR protein tyrosine kinase are therefore, expected to have great therapeutic potential in the treatment of malignant and nonmalignant epithelial diseases (38-43). These findings encourage us to synthesize novel 3, 1-benzoxazin-4-one derivatives.
General procedure for the preparation of compounds (4a, b) A mixture of (3a (44), 3b (45)) (0.01 mol) and formamide (0.015 mol) was refluxed for 3 h in boiling ethanol (30 mL), then poured into water. The precipitated solid after concentration and cooling was collected by filtration and crystallized from the proper solvent to give (4a, b). Spectroscopic data for all the compounds are given below.

General procedure for the preparation of compounds (5a, b)
A mixture of (4a, b) (0.01 mol) and chloroacetyl chloride (0.01 mol) was refluxed in boiling N, N-dimethylformamide (DMF) (30 mL) for 3 h. Then the mixture was poured into water. The precipitate was collected by filtration, dried and crystallized from the proper solvent to give (5a, b). Spectroscopic data for all the compounds are given below. taken on electro-thermal capillary melting point apparatus. Infrared spectra were recorded on a Jasco FT/IR-6100, Fourier transforms, Infrared spectrometer (Japan) at cm -1 scale using the KBr disc technique in the Central Services Laboratory, National Research Center, Dokki, Cairo, Egypt. 1 H NMR spectra were determined by using a JEOl EX-270 NMR spectrometer (Japan) at the Central Services Laboratory, National Research Center, Dokki, Cairo, Egypt. The mass spectra were measured with a Finnigan MAT SSQ-7000 mass spectrometer at the Central Services Laboratory, Cairo University, Giza, Egypt. Follow up of the reactions and checking the purity of the compounds were made by TLC on silica gelprecoated aluminum sheets (Type 60, F 254, Merck, Darmstadt, Germany) and the spots were detected by exposure to UV analysis lamp at λ 254/366 nm for few seconds.
General procedure for the preparation of compounds (3a, c, d) A solution of acid chloride (2a, c, d) (0.01 mol) and anthranilic acid (0.01 mol) in dry pyridine (30 mL) was refluxed for 3 h, the reaction mixture was cooled and poured into cold diluted HCl. The precipitate was collected by filtration and recrystallized from a proper solvent to give (3a, c, d). Spectroscopic data for all the compounds are given below. 4H-3, 1-benzoxazin-4-one (3a) Yield 80%

General procedure for the preparation of compounds (6a, b)
A mixture of (5a, b) (0.01 mol) and hydrazine hydrate (0.015 mol) was heated in boiling ethanol (30 mL) under reflux for 4 h. Then the mixture was poured into water. The precipitate was collected by filtration, dried and crystallized from the proper solvent to give (6a, b). Spectroscopic data for all the compounds are given below.

General procedure for the preparation of compounds (8c, d)
A solution of (7c, d) (44) (0.01 mol), was allowed to react with chloroacetyl chloride (0.01 mol) in refluxing pyridine about 2 h and then poured over ice/HCl. The precipitate was collected by filtration and crystallized from the proper solvent to give (8c, d). Spectroscopic data for all the compounds are given below.

General procedure for the preparation of compounds (9c, d)
A solution of compounds (7c, d) (44) (0.01 mol) and chloroacetamide (0.015 mol) was refluxed for 3 h in boiling N, N-dimethylformamide (DMF) (30 mL). Then the mixture was poured into water. The precipitate was collected by filtration, dried and crystallized from the proper solvent to give (9c, d). Spectroscopic data for all the compounds are given below.

General procedure for the preparation of compounds (10c, d)
A solution of compounds (7c, d) (44) (0.01 mol) and phenyl isothiocyanate (0.01 mol) was refluxed in boiling benzene (30 mL) for 3 h, then concentrated and crystallized from the proper solvent to give (10c, d). Spectroscopic data for all the compounds are given below.

General procedure for the preparation of compounds (11c, d)
A solution of (7c, d) (44) (0.01 mol) and benzoyl chloride (0.01 mol) in dry acetone (30 mL) was refluxed for 3 h. Excess solvent was removed and the precipitated solid obtained was crystallized from suitable solvent to obtain (11c, d). Spectroscopic data for all the compounds are given below.
Procedure: All the following procedures were done in a sterile area using a Laminar flow cabinet biosafety class II level (Baker, SG403INT, Sanford, ME, USA). Cells were suspended in RPMI 1640 medium for HePG2-MCF7 and DMEM for A549. The media are supplemented with 1% antibiotic-antimycotic mixture (10,000 U/mL Potassium Penicillin, 10,000 µg/mL Streptomycin Sulfate and 25 µg/mL Amphotericin B), 1% L-glutamine and 10% fetal bovine serum and kept at 37 °C under 5% CO 2 .
Cells were batch cultured for 10 days, then seeded at concentration of 10x10 3 cells/well in fresh complete growth medium in 96-well Microtiter plastic plates at 37 °C for 24 h under 5% CO 2 using a water jacketed Carbon dioxide incubator (Sheldon, TC2323, Cornelius, OR, USA). Media was aspirated, fresh medium (without serum) was added and cells were incubated either alone (negative control) or with different concentrations of sample to give a final concentration of (100-50-25-12.5-6.25-3.125-0.78 and 1.56 μg/mL). After 48 h of incubation, the medium was aspirated, 40 μL MTT salt (2.5 μg/mL) were added to each well and incubated for a further four hours at 37 °C under 5% CO 2 . To stop the reaction and dissolving the formed crystals, 200 μL of 10% Sodium dodecyl sulphate (SDS) in deionized water was added to each well and incubated overnight at 37 °C. A positive control which composed of 100 µg/mL was used as a known cytotoxic natural agent who gives 100% lethality under the same conditions (47, 48).
The absorbance was then measured using a microplate multi-well reader (Bio-Rad Laboratories Inc., model 3350, Hercules, California, USA) at 595 nm and a reference wavelength of 620 nm. A statistical significance was tested between samples and negative control (cells with vehicle) using independent t-test by SPSS 11 program. DMSO is the vehicle used for dissolution of plant extracts and its final concentration in the cells was less than 0.2%. The percentage of change in viability was calculated according to the formula:

General procedure for the preparation of compounds (12c, d)
A solution of (11c, d) (0.01 mol) with ammonium acetate (0.01 mol) in acetic acid (30 mL) was heated under reflux for 3 h, then poured into water. The precipitated solid after concentration and cooling was collected by filtration and crystallized from suitable solvent to give (12c, d). Spectroscopic data for all the compounds are given below. -5-(pyridin-4-yl) [1,2,4] triazolo [1, 5-c]

Cytotoxic effect on human cell line (HePG2 -MCF 7 -A549)
Cell viability was assessed by the (Reading of extract/Reading of negative control)-1) x 100. A probit analysis was carried for IC 50 and IC 90 determination using SPSS 11 program.

Generation of Ligand and Enzyme Structures
The crystal structures of EGFR (PDB code: 1M17) complex were retrieved from the RCSB Protein Data Bank (http://www.rcsb.org/pdb/ home/home.do).
We inspect the quality of the PDB file that was used using the PROSESS (Protein Structure Evaluation Suite & Server) (http:// www.prosess.com/index.php) (Figure 1, 2). In our investigation, the 3D-coordinates in X-ray crystal structure of EGFR in complex with the ligand, Erlotinib (PDB entry 1M17) was used as the receptor model in EGFR docking simulation ( Figure 3). All bound waters ligands and cofactors were removed from the protein.

Docking using Molsoft ICM 3.5-0 a program
The conversion of our PDB file into an ICM object involves the addition of hydrogen bonds, assignment of atom types and charges from the residue templates, then perform ICM small molecule docking through setup the receptor, review and adjust binding site makes receptor maps, then start docking simulation, followed by displaying the results. ICM stochastic global optimization algorithm attempts to find the global minimum of the energy function that include five grid potentials describing the interaction of the flexible ligand with the receptor and internal conformational energy of the ligand, during this process a stack of alternative low energy conformations is saved. All inhibitors were compared according to the best binding free energy (minimum) obtained among all the run.

Structure-activity relationship
The activity of the tested compounds could be correlated to structure variation and modifications. By investigating the variation in the selectivity of the tested compounds over the three cell lines, it was revealed that: (1) the activity of the designed compounds is dependent upon the substituent at the R positions. The obtained screening results showed that, nearly all of the compounds containing N-phenyl aniline showed significant inhibition for the tested three cell lines (2). Cyclization of      compound 2d afforded compound 3d (44) with the increase in activity against A549 with IC 50 values 86.1 and 77.6 µg/mL, respectively and for HePG2 with IC 50 values 75.8 and 65.1 µg /mL, respectively, while result in a little decrease in activity against MCF7 with IC 50 values 81.9 and 82.9 µg/mL, respectively (Table 2, 3, 4) (3). Compounds which have-CSNHPh group were found to be more active in the biological activities discussed in this paper than compounds which have -H. These results suggest that electron withdrawing hydrophilic      substitutes (e.g.,-CSNHPh) are more desirable for achieving the desired activity. Also Certain isothiocyanates have also been shown to bind to the mutated p53 proteins found in many types of tumors, causing an increase in the rate of cell death (4). Compounds which have CO 2 CH 2 Cl yielded the least active series of compounds in this study. Which suggests that electron withdrawing groups with lipophilic characteristics like-Cl may not be an ideal substitution to get the good activity of the designed compounds.